2. Antibodies or Immunoglobulins * Five classes of Antibodies:  IgG  IgM  IgA  IgD  IgE

4. Immunoglobulins

5. CLCL VLVL S S S S S S S S CH3CH3 CH2CH2 CH1CH1 VHVH Fc Fab F(ab) 2 Domains are folded, compact, protease resistant structures Domain Structure of Immunoglobulins Pepsin cleavage sites - 1 x (Fab) 2 & 1 x Fc Papain cleavage sites - 2 x Fab 1 x Fc Light chain C domains  or Heavy chain C domains  or 

6. CH3

7. CH2

8. CH3 CH2 CH1

9. CH3 CH2 CH1 VH1

10. CH3 CH2 CH1 VH1 VL

11. CH3 CH2 CH1 VH1 CL VL

12. CH3 CH2 CH1 VH1 CL VL

13. Hinge CH3 CH2 CH1 VH1 VL CL Elbow

14. CH3CH3 CH2CH2 Fb Fv Fb Fv Hinge Elbow CH3CH3 CH2CH2 Fb Fv Flexibility and motion of immunoglobulins

15. Hinge Fv Fb Fab CH3 CH2 CH1 VH1 VL CL Fc Elbow Carbohydrate

16. View structures

17. The Immunoglobulin Fold The characteristic structural motif of all Ig domains Barrel under construction A barrel made of a sheet of staves arranged in a folded over sheet A  barrel of 7 (C L ) or 8 (V L ) polypeptide strands connected by loops and arranged to enclose a hydrophobic interior Single V L domain

18. Unfolded V L region showing 8 antiparallel  -pleated sheets connected by loops. NH 2 COOH SS The Immunoglobulin Fold

19. Immunoglobulins must interact with a finite number of specialised molecules - Easily explained by a common Fc region irrespective of specificity - whilst simultaneously recognising an infinite array of antigenic determinants. In immunoglobulins, what is the structural basis for the infinite diversity needed to match the antigenic universe? Immunoglobulins are Bifunctional Proteins

20. Hypervariable regions Hypervariable CDRs are located on loops at the end of the Fv regions

21. Space-filling model of (Fab) 2, viewed from above, illustrating the surface location of CDR loops Light chainsGreen and brown Heavy chainsCyan and blue CDRsYellow

22. Measuring affinity of Ab to Ag Assoc between CDR and monovalent Ag can be expressed as: Ag + Ab ⇆ Ag-Ab; k 1 = forward (assoc) rate constant whereby k 1 /k -1 = K a k -1 = reverse (dissoc) rate constant the assoc/equilibrium constant K a = [Ag-Ab] value of K a depends on k 1; [Ag] [Ab] for small haptens, k 1 is high for large protein Ag ’ s, k 1 is lower

23. K a determined by equilibrium dialysis

24. Immunologic tests: 1.Precipitation Rxns: -Ab ’ s and Ag ’ s in aqueous soln ’ s form a lattice => Precipitin Lattice formation requires:1) polyvalent Ab ’ s 2) Ag must be bivalent, polyvalent Precipitation rxns, once popular, have been replaced by faster, more sensitive tests

25. Immunologic tests: 5.ELISA tests: dep on enzyme conugated to 2 Ab reacting with a specific substrate to produce a color rxn. Most sensitive of tests for Ag/Ab!! Variations of ELISA ’ s: Allows for qualitative or quantitative testing. Each one can be used for qualitative detection of Ag or Ab Also, a std curve based on known [C] ’ s of Ag/Ab can be prepped and an unknown [C} can be ascertained a.Indirect ELISA b.Sandwich ELISA c.Competitive ELISA

26. Immunologic tests: Types of ELISA ’ s…

27. Immunologic tests: 6) Western Blot Used to id specific proteins in mixtures Proteins are separated on SDS-PAGE Proteins then transferred to membrane Membrane flooded w/ radio-labelled or enz- linked poly/monoclonal Ab ’ s specific for protein

28. Immunologic tests: 7) Immunoprecipitation Provides a quick and sensitive test for finding proteins/Ag ’ s – Especially in low [C] ’ s Binds Ab to synthetic bead support  centrifuged Or 2° Ab w/ bead or magnetic bead -> collect by magnetism

29. Immunologic tests: 8) Immunofluorescence Provides a quick method for the id of pathogens and lymphocytes – Ab ’ s are conjugated with a fluorescent dye (fluorescein, rhodamine, phycoerythrin) – If Ab ’ s bind to specific Ag ’ s, they can be illum w/ UV light and emit bright colors – There are currently 2 methods employed: Direct staining Indirect staining

30. Direct and indirect Immunofluorescence